Specificity changes in the evolution of type II restriction endonucleases: a biochemical and bioinformatic analysis of restriction enzymes that recognize unrelated sequences

How restriction enzymes with their different specificities and mode of cleavage evolved has been a long standing question in evolutionary biology. We have recently shown that several Type II restriction endonucleases, namely SsoII (downward arrow CCNGG), PspGI (downward arrow CCWGG), Eco-RII (downward arrow CCWGG), NgoMIV (G downward arrow CCGGC), and Cfr10I (R downward arrow CCGGY), which recognize similar DNA sequences (as indicated, where the downward arrows denote cleavage position), share limited sequence similarity over an interrupted stretch of approximately 70 amino acid residues with MboI, a Type II restriction endonuclease from Moraxella bovis (Pingoud, V., Conzelmann, C., Kinzebach, S., Sudina, A., Metelev, V., Kubareva, E., Bujnicki, J. M., Lurz, R., Luder, G., Xu, S. Y., and Pingoud, A. (2003) J. Mol. Biol. 329, 913-929). Nevertheless, MboI has a dissimilar DNA specificity (downward arrow GATC) compared with these enzymes. In this study, we characterize MboI in detail to determine whether it utilizes a mechanism of DNA recognition similar to SsoII, PspGI, EcoRII, NgoMIV, and Cfr10I. Mutational analyses and photocross-linking experiments demonstrate that MboI exploits the stretch of approximately 70 amino acids for DNA recognition and cleavage. It is therefore likely that MboI shares a common evolutionary origin with SsoII, PspGI, EcoRII, NgoMIV, and Cfr10I. This is the first example of a relatively close evolutionary link between Type II restriction enzymes of widely different specificities.     

PspGI,a type II restriction endonuclease from the extreme thermophile Pyrococcus sp.: structural and functional studies to investigate an evolutionary relationship with several mesophilic restriction enzymes

We present here the first detailed biochemical analysis of an archaeal restriction enzyme. PspGI shows sequence similarity to SsoII, EcoRII, NgoMIV and Cfr10I, which recognize related DNA sequences. We demonstrate here that PspGI, like SsoII and unlike EcoRII or NgoMIV and Cfr10I, interacts with and cleaves DNA as a homodimer and is not stimulated by simultaneous binding to two recognition sites. PspGI and SsoII differ in their basic biochemical properties, viz. stability against chemical denaturation and proteolytic digestion, DNA binding and the pH, MgCl(2) and salt-dependence of their DNA cleavage activity. In contrast, the results of mutational analyses and cross-link experiments show that PspGI and SsoII have a very similar DNA binding site and catalytic center as NgoMIV and Cfr10I (whose crystal structures are known), and presumably also as EcoRII, in spite of the fact that these enzymes, which all recognize variants of the sequence -/CC-GG- (/ denotes the site of cleavage), are representatives of different subgroups of type II restriction endonucleases. A sequence comparison of all known restriction endonuclease sequences, furthermore, suggests that several enzymes recognizing other DNA sequences also share amino acid sequence similarities with PspGI, SsoII and EcoRII in the region of the presumptive active site. These results are discussed in an evolutionary context.     

Evolutionary relationship between different subgroups of restriction endonucleases

The type II restriction endonuclease SsoII shows sequence similarity with 10 other restriction endonucleases, among them the type IIE restriction endonuclease EcoRII, which requires binding to an effector site for efficient DNA cleavage, and the type IIF restriction endonuclease NgoMIV, which is active as a homotetramer and cleaves DNA with two recognition sites in a concerted reaction. We show here that SsoII is an orthodox type II enzyme, which is active as a homodimer and does not require activation by binding to an effector site. Nevertheless, it shares with EcoRII and NgoMIV a very similar DNA-binding site and catalytic center as shown here by a mutational analysis, indicative of an evolutionary relationship between these three enzymes. We suggest that a similar relationship exists between other orthodox type II, type IIE, and type IIF restriction endonucleases. This may explain why similarities may be more pronounced between members of different subtypes of restriction enzymes than among the members of a given subtype.     

Cleavage of synthetic substrates containing non-nucleotide inserts by restriction endonucleases. Change in the cleavage specificity of endonuclease SsoII.

A study was made of the interaction between restriction endonucleases recognizing CCNGG (SsoII and ScrFI) or CCA/TGG (MvaI and EcoRII) DNA sequences and a set of synthetic substrates containing 1,3-propanediol, 1,2-dideoxy-D-ribofuranose or 9-[1'-hydroxy-2'-(hydroxymethyl)ethoxy] methylguanine (gIG) residues replacing either one of the central nucleosides or dG residues in the recognition site. The non-nucleotide inserts (except for gIG) introduced into the recognition site both increase the efficiency of SsoII and change its specificity. A cleavage at the noncanonical position takes place, in some cases in addition to the correct ones. Noncanonical hydrolysis by SsoII occurs at the phosphodiester bond adjacent to the point of modification towards the 5'-end. With the guanine base returned (the substrate with gIG), the correct cleavage position is restored. ScrFI specifically cleaves all the modified substrates. DNA duplexes with non-nucleotide inserts (except for the gIG-containing duplex) are resistant to hydrolysis by MvaI and EcoRII. Prompted by the data obtained we discuss the peculiarities of recognition by restriction endonucleases of 5-membered DNA sequences which have completely or partially degenerated central base pairs. It is suggested that SsoII forms a complex with DNA in an 'open' form.     

PsiI, a novel restriction endonuclease recognizing the DNA sequence 5'-TTA downward arrow TAA-3'

PsiI, a novel restriction endonuclease produced by the bacterial strain Pseudomonas sp. SE-G49, has been isolated and characterized. The enzyme cleaves DNA in the middle of its palindromic recognition sequence 5'-TTA downward arrow TAA-3'. Thus, PsiI belongs to a rare group of type II restriction endonucleases whose recognition sites consist of AT base pairs only.     

Probing the dynamics of restriction endonuclease NgoMIV‐DNA interaction by single‐molecule FRET

Many type II restriction endonucleases require two copies of their recognition sequence for optimal activity. Concomitant binding of two DNA sites by such an enzyme produces a DNA loop. Here we exploit single‐molecule Förster resonance energy transfer (smFRET) of surface‐immobilized DNA fragments to study the dynamics of DNA looping induced by tetrameric endonuclease NgoMIV. We have employed a DNA fragment with two NgoMIV recognition sites and a FRET dye pair such that upon protein‐induced DNA looping the dyes are brought to close proximity resulting in a FRET signal. The dynamics of DNA ‐ NgoMIV interactions proved to be heterogeneous, with individual smFRET trajectories exhibiting broadly different average looped state durations. Distinct types of the dynamics were attributed to different types of DNA ‐ protein complexes, mediated either by one NgoMIV tetramer simultaneously bound to two specific sites (“slow” trajectories) or by semi‐specific interactions of two DNA‐bound NgoMIV tetramers (“fast” trajectories), as well as to conformational heterogeneity of individual NgoMIV molecules.     

DNA cleavage reactions by type II restriction enzymes that require two copies of their recognition sites

Several type II restriction endonucleases interact with two copies of their target sequence before they cleave DNA. Three such enzymes, NgoMIV, Cfr10I and NaeI, were tested on plasmids with one or two copies of their recognition sites, and on catenanes containing two interlinked rings of DNA with one site in each ring. The enzymes showed distinct patterns of behaviour. NgoMIV and NaeI cleaved the plasmid with two sites faster than that with one site and the catenanes at an intermediate rate, while Cfr10I gave similar steady-state rates on all three substrates. Both Cfr10I and NgoMIV converted the majority of the substrates with two sites directly to the products cut at both sites, while NaeI cleaved just one site at a time. All three enzymes thus synapse two DNA sites through three-dimensional space before cleaving DNA. With Cfr10I and NgoMIV, both sites are cleaved in one turnover, in a manner consistent with their tetrameric structures, while the cleavage of a single site by NaeI indicates that the second site acts not as a substrate but as an activator, as reported previously. The complexes spanning two sites have longer lifetimes on catenanes with one site in each ring than on circular DNA with two sites, which indicates that the catenanes have more freedom for site juxtaposition than plasmids with sites in cis.     

Affinity Modification of the Restriction Endonuclease <Emphasis Type="Italic">Sso</Emphasis>II by 2′-Aldehyde-Containing Double Stranded DNAs

Properties of 2'-aldehyde-containing double stranded DNAs (dsDNAs) have been studied for the first time as substrate analogs of the restriction endonuclease SsoII. These reactive oligonucleotides were successfully cross-linked to the restriction endonuclease SsoII by reductive amination, and conditions for DNA-protein conjugate trypsinolysis followed by the oligonucleotide-peptide conjugate purification were optimized. Use of MALDI-TOF mass spectrometry revealed that covalent linkage forms between the sugar moiety of the central pyrimidine nucleoside of the SsoII recognition site and Lys173 of the enzyme. The latter is probably involved in initial steps of enzyme-substrate recognition during dsDNA readout.     

Cytosine DNA-methylase of a SsoII-type from Shigella sonnei 47 cells

Shigella sonnei 47 cells were found to contain DNA-methylase SsoII which is a modifying component of the system of host specificity of SsoII. The recognition sequence (RS) of methylase SsoII is represented by a five-member palyndromic structure--5'...CCNGG...3'--with a degenerated central nucleotide. Modification of SsoII affords protection of acceptor DNA not only from SsoII type restriction, but also from other restrictases, e. g., Eco RII having an analogous RS but with a less degenerated central nucleotide pair. A simple and rapid procedure for isolation and purification of DNA-methylase ScoII, which employs hydrophobic chromatography on phenyl-Sepharose, has been developed. The enzyme preparation does not contain trace amounts of specific and nonspecific endonucleases and keeps stable on storage in 30% glycerol over a period of one year.     

Iterative database searches demonstrate that glycoside hydrolase families 27, 31, 36 and 66 share a common evolutionary origin with family 13

A catalytic sequence motif PDX10-30(E/D)XK is found in many restriction enzymes. On the basis of sequence similarities and mapping of the conserved residues to the crystal structure of NgoMIV we suggest that residues D160, K182, R186, R188 and E195 contribute to the catalytic/DNA binding site of the Ecl18kI restriction endonuclease. Mutational analysis confirms the functional significance of the conserved residues of Ecl18kI. Therefore, we conclude that the active site motif 159VDX21KX12E of Ecl18kI differs from the canonical PDX10-30(E/D)XK motif characteristic for most of the restriction enzymes. Moreover, we propose that two subfamilies of endonucleases Ecl18kI/PspGI/EcoRII and Cfr10I/Bse634I/NgoMIV, specific, respectively, for CCNGG/CCWGG and RCCGGY/GCCGGC sites, share conserved active site architecture and DNA binding elements.     

The interaction of DNA duplexes containing 2-aminopurine with restriction endonucleases EcoRII and SsoII.

Oligonucleotides containing 2-aminopurine (2-AP) in place of G or A in the recognition site of EcoRII (CCT/AGG) or SsoII (CCNGG) restriction endonucleases have been synthesized in order to investigate the specific interaction of DNA with these enzymes. Physicochemical properties (CD spectra and melting behaviour) have shown that DNA duplexes containing 2-aminopurine exist largely in a stable B-like form. 2-Aminopurine base paired with cytidine, however, essentially influences the helix structure. The presence of a 2-AP-C mismatch strongly reduces the stability of the duplexes in comparison with the natural double strand, indicated by a biphasic melting behaviour. SsoII restriction endonuclease recognizes and cleaves the modified substrate with a 2-AP-T mismatch in the centre of the recognition site, but it does not cleave the duplexes containing 2-aminopurine in place of inner and outer G, or both. EcoRII restriction endonuclease does not cleave duplexes containing 2-aminopurine at all. The two-substrate mechanism of EcoRII-DNA interaction, however, allows hydrolysis of the duplex containing 2-aminopurine in place of adenine in the presence of the canonical substrate.     

Synthesis and characteristics of modified DNA fragments containing thymidine glycol residues

Chemical synthesis of a series of modified oligodeoxyribonucleotides containing one or two residues of thymidine glycol (5,6-dihydro-5,6-dihydroxythymidine), the main product of oxidative DNA damage, is described. The thermal stability of DNA duplexes containing thymidine glycol residues was studied using UV spectroscopy. Introduction of even one thymidine glycol residue into the duplex structure was shown to result in its significant destabilization. Data on the interaction of DNA methyltransferases and type II restriction endonucleases with DNA ligands containing oxidized thymine were obtained for the first time. Introduction of a thymidine glycol residue in the central degenerate position of the recognition site of restriction endonuclease SsoII was found to result in an increase in the initial hydrolysis rate of the modified duplex in comparison with that of unmodified structure. The affinity of C5-cytosine methyltransferase SsoII for the DNA duplex bearing thymidine glycol was found to be twofold higher than for the unmodified substrate. However, such a modification of the DNA ligand prevents its methylation.     

Isolation and characterization of a new poly(3-hydroxybutyrate)-degrading, denitrifying bacterium from activated sludge

One hundred and forty isolates of thermophilic bacteria from the genus Thermus were screened for the presence of restriction endonuclease activity. Thermostable isoschizomers of restriction endonucleases, such as AceIII, BbvI, BglI, BsePI, FnuDII, HgiAI, MaeII, MboI, MseI, PvuII, StuI, TaqI, Tsp4CI, TspEI, XhoI and XmaIII, were isolated. Two restriction enzymes, TatI and TauI, recognizing novel degenerate sequences 5'-W (downward arrow)GTACW-3' and 5'-GCSG (downward arrow)C-3' respectively were partially purified and the recognition and cleavage sites were determined.     

Cleavage of individual DNA strands by the different subunits of the heterodimeric restriction endonuclease BbvCI

BbvCI cleaves an asymmetric DNA sequence, 5'-CC downward arrow TCAGC-3'/5'-GC downward arrow TGAGG-3', as indicated. While many Type II restriction enzymes consist of identical subunits, BbvCI has two different subunits: R(1), which acts at GC downward arrow TGAGG; and R(2), which acts at CC downward arrow TCAGC. Some mutants of BbvCI with defects in one subunit, either R(1)(-)R(2)(+) or R(1)(+)R(2)(-), cleave only one strand, that attacked by the native subunit. In analytical ultracentrifugation at various concentrations of protein, wild-type and mutant BbvCI enzymes aggregated extensively, but are R(1)R(2) heterodimers at the concentrations used in DNA cleavage reactions. On a plasmid with one recognition site, wild-type BbvCI cleaved both strands before dissociating from the DNA, while the R(1)(-)R(2)(+) and R(1)(+)R(2)(-) mutants acted almost exclusively on their specified strands, albeit at relatively slow rates. During the wild-type reaction, the DNA is cleaved initially in one strand, mainly that targeted by the R(1) subunit. The other strand is then cleaved slowly by R(2) before the enzyme dissociates from the DNA. Hence, the nicked form accumulates as a transient intermediate. This behaviour differs from that of many other restriction enzymes, which cut both strands at equal rates. However, the activities of the R(1)(+) and R(2)(+) subunits in the wild-type enzyme can differ from their activities in the R(1)(+)R(2)(-) and R(1)(-)R(2)(+) mutants. Each active site in BbvCI therefore influences the other.     

Synthesis and characteristics of modified DNA fragments containing thymidine glycol residues

Chemical synthesis of a series of modified oligodeoxyribonucleotides containing one or two residues of thymidine glycol (5,6-dihydro-5,6-dihydroxythymidine), the main product of oxidative DNA damage, is described. The thermal stability of DNA duplexes containing thymidine glycol residues was studied using UV spectroscopy. Introduction of even one thymidine glycol residue into the duplex structure was shown to result in its significant destabilization. Data on the interaction of DNA methyltransferases and type II restriction endonucleases with DNA ligands containing oxidized thymine were obtained for the first time. Introduction of a thymidine glycol residue into the central degenerate position of the recognition site of restriction endonuclease SsoII was found to result in an increase in the initial hydrolysis rate of the modified duplex in comparison with that of the unmodified structure. The affinity of C5-cytosine methyltransferase SsoII for the DNA duplex bearing thymidine glycol was found to be twofold higher than for the unmodified substrate. However, such a modification of the DNA ligand prevents its methylation. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2008, vol. 34, no. 2; see also http://www.maik.ru.     

Analysis of DNA looping interactions by type II restriction enzymes that require two copies of their recognition sites

Before cleaving DNA substrates with two recognition sites, the Cfr10I, NgoMIV, NaeI and SfiI restriction endonucleases bridge the two sites through 3D space, looping out the intervening DNA. To characterise their looping interactions, the enzymes were added to plasmids with two recognition sites interspersed with two res sites for site-specific recombination by Tn21 resolvase, in buffers that contained either EDTA or CaCl2 so as to preclude DNA cleavage by the endonuclease; the extent to which the res sites were sequestered into separate loops was evaluated from the degree of inhibition of resolvase. With Cfr10I, a looped complex was detected in the presence but not in the absence of Ca(2+); it had a lifetime of about 90 seconds. Neither NgoMIV nor NaeI gave looped complexes of sufficient stability to be detected by this method. In contrast, SfiI with Ca(2+) produced a looped complex that survived for more than seven hours, whereas its looping interaction in EDTA lasts for about four minutes. When resolvase was added to a SfiI binding reaction in EDTA followed immediately by CaCl2, the looped DNA was blocked from recombination while the unlooped DNA underwent recombination. By measuring the distribution between looped and unlooped DNA at various SfiI concentrations, and by fitting the data to a model for DNA binding by a tetrameric protein to two sites in cis, an equilibrium constant for the looping interaction was determined. The equilibrium constant was essentially independent of the length of DNA between the SfiI sites.     

A view of consecutive binding events from structures of tetrameric endonuclease SfiI bound to DNA

Many reactions in cells proceed via the sequestration of two DNA molecules in a synaptic complex. SfiI is a member of a growing family of restriction enzymes that can bind and cleave two DNA sites simultaneously. We present here the structures of tetrameric SfiI in complex with cognate DNA. The structures reveal two different binding states of SfiI: one with both DNA-binding sites fully occupied and the other with fully and partially occupied sites. These two states provide details on how SfiI recognizes and cleaves its target DNA sites, and gives insight into sequential binding events. The SfiI recognition sequence (GGCCNNNN[downward arrow]NGGCC) is a subset of the recognition sequence of BglI (GCCNNNN[downward arrow]NGGC), and both enzymes cleave their target DNAs to leave 3-base 3' overhangs. We show that even though SfiI is a tetramer and BglI is a dimer, and there is little sequence similarity between the two enzymes, their modes of DNA recognition are unusually similar.     

DNA-methyltransferase SsoII as a bifunctional protein: Features of the interaction with the promoter region of SsoII restriction-modification genes

DNA duplexes bearing an aldehyde group at the 2'-position of the sugar moiety were used for affinity modification of (cytosine-5)-DNA methyltransferase SsoII. It is shown that lysine residues of M.SsoII N-terminal region are located in proximity to DNA sugar-phosphate backbone of a regulatory sequence of promoter region of SsoII restriction-modification enzyme coding genes. The ability of the two M.SsoII subunits to interact with DNA regulatory sequence has been demonstrated by affinity modification using DNA duplexes with two 2'-aldehyde groups. Changes in nucleotide sequence of one half of the regulatory region prevented cross-linking of the second M.SsoII subunit. The results on sequential affinity modification of M.SsoII by two types of modified DNA ligands (i.e. by 2'-aldehyde-containing and phosphoryldisulfide-containing) have demonstrated the possibility of covalent attachment of the protein to two different DNA recognition sites: regulatory sequence and methylation site.     

Restriction endonucleases and their applications

Restriction endonucleases are deoxyribonucleases which cleave double-stranded DNA into fragments. With only one exception, all restriction endonucleases recognize short, non-methylated DNA sequences. Restriction endonucleases can be divided into two groups based on the position of the cleavage site relative to the recognition sequence. Class I restriction endonucleases cleave double-stranded DNA at positions outside the recognition sequence and generate fragments of random size. The cleavage sites of Class II restriction endonucleases are located, in most cases, within the recognition sequence. Most of the Class II restriction endonucleases recognize 4, 5, or 6 base pair palindromes and generate fragments with either flush ends or staggered ends. DNA fragments with staggered ends contain 3, 4, or 5 nucleotide single-stranded tails called ‘sticky ends’. DNA fragments produced by Class II restriction endonuclease cleavage can be separated on gels according to their molecular weight. The fragments can be isolated from the gel and used for sequence analysis to elucidate genetic information stored in DNA. Further, an isolated fragment can be inserted into a small extrachromosomal DNA, e.g. plasmid, phage or viral DNA, and its replication and expression can be studied in clones of prokaryotic or eukaryotic cells. Restriction endonucleases and cloning technology are powerful modern tools for attacking genetic problems in medicine, agriculture and industrial microbiology.